B American Society for Mass Spectrometry, 2015DOI: 10.1007/s13361-015-1224-9J. Am. Soc. Mass Spectrom. (2015) 26:1702Y1712

RESEARCH ARTICLE

Paper-Based Electrochemical Cell Coupled to MassSpectrometry

Yao-Min Liu, Richard H. PerryDepartment of Chemistry, University of Illinois, Urbana, IL 61801, USA

Abstract. On-line coupling of electrochemistry (EC) to mass spectrometry (MS) is apowerful approach for identifying intermediates and products of EC reactions in situ.In addition, EC transformations have been used to increase ionization efficiency andderivatize analytes prior to MS, improving sensitivity and chemical specificity. Re-cently, there has been significant interest in developing paper-based electroanalyticaldevices as they offer convenience, low cost, versatility, and simplicity. This reportdescribes the development of tubular and planar paper-based electrochemical cells(P-EC) coupled to sonic spray ionization (SSI) mass spectrometry (P-EC/SSI-MS).The EC cells are composed of paper sandwiched between two mesh stainless steelelectrodes. Analytes and reagents can be added directly to the paper substrate along

with electrolyte, or delivered via the SSI microdroplet spray. The EC cells are decoupled from the SSI source,allowing independent control of electrical and chemical parameters. We utilized P-EC/SSI-MS to characterizevarious EC reactions such as oxidations of cysteine, dopamine, polycyclic aromatic hydrocarbons, and diphenylsulfide. Our results show that P-EC/SSI-MS has the ability to increase ionization efficiency, to perform online ECtransformations, and to capture intermediates of EC reactions with a response time on the order of hundreds ofmilliseconds. The short response time allowed detection of a deprotonated diphenyl sulfide intermediate, whichexperimentally confirms a previously proposed mechanism for EC oxidation of diphenyl sulfide to pseudodimersulfonium ion. This report introduces paper-based EC/MS via development of two device configurations (tubularand planar electrodes), as well as discusses the capabilities, performance, and limitations of the technique.Keywords: Paper analytical device, Electrochemistry, Mass spectrometry, Ambient, Sonic spray ionization,Polycyclic aromatic hydrocarbons, Electrolytic cell, Electrochemical reaction mechanisms, Orbitrap, Onlineelectrochemistry, Solid-contact electrodes

Received: 8 May 2015/Revised: 15 June 2015/Accepted: 17 June 2015/Published Online: 27 August 2015

Introduction

In recent years, there has been significant interest in devel-oping paper-based devices for analytical and biosensor ap-

plications because they are cheap, malleable, disposable, easy-to-use, and simple [1]. Paper-based electroanalytical devicesoffer the potential for high sensitivity and low limits of detec-tion [1]. The first paper-based electrochemical (EC) device wasintroduced by Henry and coworkers [2] in 2009 for the detec-tion of glucose, lactate, and uric acid in biological samples.This device utilized a carbon ink/Prussian blue mixture for the

working electrode (WE) and counter electrode (CE), and silver/silver chloride ink for the reference electrode (RE). Cui et al.[3] developed a paper EC sensor comprised of solid-contact ionsensing and reference electrodes to determine the concentrationof potassium ions in samples absorbed into a paper substrate(one end of the paper substrate was immersed in the samplesolution), demonstrating the ability to integrate solid-contactelectrodes with the convenience of disposable paper substrates[3]. Online coupling of paper-based EC cells to mass spectrom-etry (MS) would open for study a new set of analytical tech-niques and applications. However, to our knowledge, thistechnological advance has not been demonstrated.

Electrochemistry coupled to MS (EC/MS) was first intro-duced by Bruckenstein and Gadde in 1971 [4]. Since then, EC/MS has found wide application in many areas of science andindustry, such as the characterization of EC processes [5], drugmetabolism [6], and biomolecules (e.g., proteins [7–12] and

Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1224-9) contains supplementary material, which is availableto authorized users.

Correspondence to: Richard Perry; e-mail: rhperry@illinois.edu

deoxyribonucleic acid [13, 14]), as well as online derivatizationof functional groups [15, 16] and chemical imaging [5, 17, 18]. InEC/MS, the mass spectrometer serves as a sensitive detector foridentifying products and intermediates generated in EC process-es, providing mass-to-charge (m/z) ratios and structural informa-tion via tandemMS spectrometry (MS/MS) [5]. Conversely, ECtransformations prior to MS can selectively transform analytes toimprove ionization efficiency and selectivity [5, 19].

EC is typically coupled to electrospray ionization (ESI) be-cause of its high sensitivity and its ability to transfer intact non-volatile species from solution to the gas phase (viz. ESI is a ‘soft’ionization method) [20–23]. In ESI, EC processes occur at thesolution-capillary interface (‘solution’ in this context refers to thesolvent carrying analytes and reactants through the EC cell andESI emitter) [24–31], which are influenced by the ESI current,capillary material, and composition of the solution. As a result ofthis inherent complexity in ESI, a distinct EC cell is desired toenable independent physical, electrical, and chemical control[32–35]. Two-electrode [34, 36–41] and three-electrode [33,35, 42–44] EC/ESI-MS configurations have been developed thatachieve electrical decoupling through (a) floating the EC cell onthe potential induced by the ESI high voltage or (b) electricallyisolating the electronic circuits. An interesting example is anintegrated three-electrode EC/ESI-MS device developed by Coleand coworkers [33, 35, 42] in which solution interacts with theECworking electrode a fewmillimeters before the end of the ESIsource, leading to shorter response times (tr < 3 s; time betweenEC conversion and detection at three times the signal-to-noise (S/N)) compared to typical configurations that have tubingconnecting the EC cell and ESI emitter (tr is determined by tubinglength, tubing internal diameter, and solution flow rate).

The field of ambient mass spectrometry (AMS) was startedby Cooks and coworkers with the development of desorptionelectrospray ionization (DESI). AMS provides the ability toperform chemical analyses of systems in their natural state,which has led to impactful discoveries in numerous researchareas such as forensics, environmental science, and cancer bio-chemistry [45–53]. Recently, Chen and coworkers [11, 54–58]demonstrated that DESI can characterize liquid samples (liquidsampling DESI; LS-DESI) such as the effluent from chromato-graphic columns [59–61] and EC flow-cells [10, 62–64]. Whenmicrodroplets from a LS-DESI source impact the outlet oftubular [54] or thin layer [10] EC flow cells, species in the ECeffluent are desorbed into secondary microdroplets that traveltowards a MS for detection. The electrical, chemical, and phys-ical components of the EC cell and LS-DESI source [54] areseparate, which enables independent control of electrical poten-tials, minimizes deleterious effects of electrolytes on ionizationefficiency, prevents unwanted reactions that can potentially oc-cur at ESI electrode surfaces, increases the scope of EC chemicalsystems available for study by EC/MS, and reduces carry-overeffects in the ionization source. Recently, the Zare and Chenlaboratories developed an EC/DESI-MS configuration that en-ables ambient capture of solution-phase intermediates with life-times of tens of milliseconds [65], which has significant advan-tage for elucidating EC mechanisms. In another significant

advance, the Cooks and Ouyang laboratories introduced a pa-per-based spray ionization source (PSI) that provides an elegant,simple, portable, and cheap method for analyzing various sam-ples (e.g. chemicals and cells) and reactions by MS [66–74].Coupling P-EC to MS would leverage the advantages of paper(e.g. cheap, versatile, and disposable), as well as advance paperelectroanalytical technologies through providing mechanisticinformation about processes occurring in paper.

Herein, we harness the benefits of paper for EC/MS applica-tions. We report the development and characterization of a two-electrode paper-based EC cell for coupling to MS (hereafterreferred to as P-EC/MS; Figure 1). The P-EC/MS system iscomposed of (a) a paper substrate sandwiched between two sol-id-contact stainless steel (SS) mesh electrodes (planar P-EC) and(b) an ambientmicrodroplet probe directed at P-EC in linewith theMS (Figures 1a and 1c). In a typical experiment, primary

15 mm2 mm

MS

++++ ++ ++++ ++++

+++

+ +++

++

+ +

++

+

SSIPure solvent/analyte

Liquid: 20 L/minN2 = 200 PSI

(a)

Electrode 1 (E1) Electrode 2 (E2)

Impact region on the paper substrate containing analyte and/or electrolyteV

DC Power Supply ( V = 4 V)

+-

(b)

MS

++++ ++ ++++ ++++

+++

+ +++

++

+ +

++

+

+- V

(c) P-EC Assembly

MS

Microdroplet Emitter (SSI)

Paper substrate

Alligator clip

Interaction with analyte/reagents in paper

(

E3: 15 mm

E4: 12 mm

2 mm 2 mm

x

y

z

z

y

z

y

Binder clip and insulator

)green

Figure 1. Schematic of a planar (a) and tubular (b) paper-based EC assembly (P-EC) coupled to MS. (c) Photographshowing the actual planar P-EC/MS experimental setup.Figures are not drawn to scale

Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry 1703

microdroplets containing reagents/analytes impact the paper sub-strate saturated with electrolyte after passing through SS meshelectrode E1. It is also possible to use pure solvents in the SSImicrodroplet spray and saturate the paper with analytes/reagentsand electrolyte. However, unless stated otherwise, the capabilitiesof P-EC/MS will be described with reagents/analytes added to themicrodroplet spray. Application of a potential across the electrodes(ΔV) produces EC intermediates and products, which are extractedinto secondary microdroplets that travel through E2 and undergosonic spray ionization (SSI) [75] to generate gas-phase ions. Theflexibility of the SS mesh electrodes and paper substrate make theEC cell malleable, providing multiple configurations for couplingP-EC cells to MS (Figure 1b shows the tubular configuration).

The capabilities of P-EC/SSI-MS were demonstrated usingwell-known EC reactions such as oxidation of cysteine (Cys),diphenyl sulfide (PhSPh), and polycyclic aromatic hydrocarbons(PAH). P-EC/SSI-MS produce results similar to previously pub-lished EC/MS characterization of these reactions, demonstratingits ability to monitor EC processes, to increase ionization efficien-cy of nonpolar compounds, to perform online derivatization ofspecific analytes, and to distinguish isomers based on differing ECproperties. One particularly exciting observation is that P-EC/SSI-MS has a short tr, which facilitates detection of transient interme-diates generated at the electrode surfaces on the hundreds ofmilliseconds time scale. The shorter tr of P-EC/SSI-MS alloweddetection of a deprotonated diphenyl sulfide species ([PhSC6H4])during oxidation [42], providing evidence for a mechanism in-volving a cation or radical [Ph-S-C6H4] species to formpseudodimer sulfonium ion product [Ph2S

+C6H4SPh]. In addi-tion, the P-EC cell and SSI source are discrete systems, allowingindependent control of electrical and chemical parameters. Finally,using a paper substrate between the electrodes provides a simple,cheap, disposable, and versatile approach for coupling EC to MS.

ExperimentalMaterials

All chemicals were purchased from Sigma-Aldrich (St. Louis,MO, USA) and used without further purification. The stainlesssteel meshes (304 SS woven mesh with an unpolished (mill)finish; wire diameter = 200 μm; 30% open area) were pur-chased from Amazon.com (Seattle, WA, USA). The papersubstrate was made from two sheets of Kimwipe (Kimberly-Clark, Neenah, WI, USA) and purchased from Thermo FisherScientific (San Jose, CA, USA). The direct current (DC) powersupply (Keysight U8002A; 0–30 V; 0–5 A) was purchasedfrom Testequity, LLC (Moorpark, CA, USA).

Design and Operation of the Paper-BasedElectrochemical Cells

In planar P-EC, a paper substrate is placed between and incontact with two SS mesh electrodes (E1 and E2 in Figure 1a;E1: x × y × z (thickness) = 16mm × 11mm× 200 μm; E2: x × y× z = 20 mm × 15 mm × 200 μm). The dimensions of E1 < E2

and of the paper substrate > E2 to minimize the possibility ofelectrical shorting during operation of the EC cell. The x and ydimensions of the paper substrate are relatively similar to theelectrode E1 (paper thickness = ~50 μm per sheet) and the E1-paper-E2 components of the P-EC assembly (500 μm totalthickness) are held together with two regular office metalbinder clips. Plastic insulators (colored green in Figure 1c)were inserted between the binder clips and the electrode sur-faces. The P-EC assembly was positioned at 90o relative to theMS inlet and held in place using three-pronged clamps attachedto ring stands (Figure 1c). The microdroplet SSI emitter wasplaced 17 mm from the MS inlet (0o relative) and 2 mm fromE1. In the tubular P-EC (Figure 1b) configuration, two sheets ofSS mesh (dimensions of E4 are less than E3) separated by apaper substrate of relatively similar dimensions were wrappedaround a circular object to produce tubular electrodes (internaldiameter (i.d.) × outer diameter (o.d.) × z = 3.2 mm × 3.8 mm ×15 mm; internal surface area (ISA) = [π × (ID/2)2 × z] =120.6 mm2). The microdroplet emitter is placed 0o relative tothe MS inlet, and the emitter-to-P-EC and P-EC-to-inlet dis-tances are 2 mm (Figure 1b). For both the planar and tubular P-EC assemblies, alligator clips were used to connect each elec-trode to terminals of the DC power supply (Figure 1).

Construction of the microdroplet sprayer has been previouslydescribed by Cooks and coworkers [53]. Briefly, the sprayerconsisted of a Swagelok (Swagelok, Fremont, CA, USA) 1/16′′SS tee through which a FS capillary (250 μm i.d.; 360 μm o.d.);Polymicro Technologies, Lisle, IL, USA) delivered liquid from aHarvard Apparatus Standard Pump 22 (Holliston, MA, USA)syringe pump. FS passed through both 180o openings, and washeld in placewith a SS ferrule and SS nut at the end of the tee. Theother end of FS passed through a SS capillary (length = 5 cm; i.d.= 0.020′′), which was held in place with a second SS ferrule andSS nut. FS capillary protruded from the end of the SS capillary by~1 mm. Different solution compositions were sprayed with flowrate at 20 μL/min. Sheath gas (nitrogen; N2) entered the sprayer atthe 90o opening of the tee (line pressure = ~200 PSI) and exitedSS capillary, generating the microdroplet spray.

In a typical P-EC/SSI-MS experiment, the paper substrate issaturated with electrolyte using a Pasteur pipet and kept satu-rated throughout the analysis. Then, 4 V is supplied to theelectrodes using a DC power supply [for the planar configura-tion, the positive electrode (E2) was closest to the MS; fortubular P-EC, the positive electrode (E3) forms the internalsurface of the assembly]. Analyte is then delivered to the P-ECassembly via the microdroplet spray emitter. In the planarconfiguration, microdroplets impact the paper substrate afterpassing through E1. Electrochemical species generated in thepaper substrate are extracted by subsequent impactingmicrodroplets, which carry the analytes to the mass spectrom-eter. In tubular P-EC/MS, the primary microdroplets deliver theanalyte to the paper substrate after passing through E3(Figure 1b). Generated electrochemical species are thendesorbed and extracted into secondary microdroplets. An im-portant difference between the planar and tubular designs isthat the secondary microdroplets can either travel to the mass

1704 Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry

spectrometer or undergo subsequent collisions with the papersubstrate leading to additional reactions. As a result, the lengthand ISA of the tubular electrodes influence parameters such asthe reaction time, ionization efficiency, and sensitivity.

Mass Spectrometry

The microdroplets entering the mass spectrometer undergodesolvation using a capillary temperature of 275oC on anLTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientif-ic, San Jose, CA, USA) [76–81]. Unless specified otherwise, theOrbitrap MS was typically operated using the following param-eters: single-stage m/z range = m/z 60–500, m/z resolutionsetting = 100,000 at m/z 400, mass accuracy = 2–5 ppm,microscans = 1, ion injection time = 500 ms, tube lens voltage= 110 V, spray voltage = 0 kV (SSI). For experiments thatmeasure tr, ion detection was performed using the linear ion trapof the hybrid mass spectrometer (ITMS) with similar settings asdescribed above, except that ion injection time = 100 ms.

Results and DiscussionCharacterization of Planar P-EC/SSI-MS

The well-known EC formation of disulfides from thiols (e.g.,Cys in Figure 2a) [10] was used to characterize features of

planar P-EC/SSI-MS (Figure 1a). SSI was used to avoidvoltage-induced reaction pathways such as the formation ofreactive oxygen species, which can occur with water (H2O) oracetonitrile (CH3CN) as ESI solvents [82]. When a solution ofCys (MW = 121 gmol–1; 10 mM in 1:1 water:methanol(H2O:CH3OH)) was used as the microdroplet spray and KCl(10 mM) as the electrolyte added to the paper substrate, appli-cation of a potential difference (ΔV) = 4 V across electrodes E1and E2 yielded mass spectra (Figure 2b and c) showing ionsignals corresponding to [Cys + H]+ (m/z 122.026), [Cys +Na]+ (m/z 144.008), [Cys + 2Na – H]+ (m/z 165.990), [Cys-S-S-Cys + H]+ (m/z 241.030), [Cys-S-S-Cys + Na]+ (m/z263.012), and [Cys-S-S-Cys + 2Na – H]+ (m/z 284.994). Theion signals corresponding to the disulfide product were notobserved when ΔV = 0 V (Figure 2c), successfully demonstrat-ing P-EC oxidation followed by online SSI-MS.

The paper substrate is sufficiently porous such that ionsignal intensities are not significantly affected when the ECcell is placed between the SSI and the MS inlet. When varioussolutions of Cys (10–4 M to 10–2 M in increments of 5 × 10–4 M) were directed at the mass spectrometer inlet capillary, thesum of the absolute intensities of Cys ionic species ([Cys + H]+

(m/z 122.026), [Cys + Na]+ (m/z 144.008), and [Cys + 2Na –H]+ (m/z 165.990) represented as Σ[M + n]+, where M is theneutral molecule and n represents H, Na, or (2Na – H)) at eachconcentration was relatively similar with and without the ECcell (Figure 3a; [KCl] = 0 M; ΔV = 0 V). In addition, applica-tion of 4 V to the EC cell did not significantly change Σ[Cys +n]+ (Figure S1a). Unless stated otherwise, error bars representthe standard deviation of three values and a fresh paper sub-strate was used for each measurement. In addition, it is impor-tant to note that the position of P-EC (angles and distances) hasa significant effect on ion signal intensity, so the same param-eters were used for all measurements. The ratio (Σ[Cys + n]+

with P-EC)/(Σ[Cys + n]+ without P-EC) across all Cys con-centrations = 1.1 ± 0.3, which indicates that the paper EC celldoes not significantly affect sampling efficiency (SE) = iontransmission efficiency (TE) + ionization efficiency (IE) whenplaced between the emitter and the MS inlet.

The concentration of KCl affects ionization efficiency (IE)from the paper substrate, whereasΔV has no effect on IE. Whenthe paper substrate was saturated with various concentrationsof KCl (1, 10, and 100 mM) atΔV= 0 V, the ratio (Σ[Cys + n]+

for [KCl] = 100 mM)/(Σ[Cys + n]+ for [KCl] = 1 mM) = 3.5 ±0.5 for [Cys] = 10 mM (Figure 3b), indicating a slight increasein ion signal intensities. When the KCl(aq) concentration isgreater than 10 mM, KCl(s) gradually accumulates on the MSinlet, eventually obstructing ion transfer into the mass spec-trometer. For this reason, the remaining P-EC/MS experimentswere performed at [KCl] = 10 mM. When ΔV was increasedfrom 0 to 5 V in 1 V increments while holding the KCl and Cysconcentrations constant, Σ[Cys + n]+ = (7.0 ± 0.6) × 104 withrelative standard deviation (RSD) of only 9% (Figure S1b). Allthese results show that (1) potentials applied to the EC cell (0–7 V) have no observable effect on the average charge of thesecondary microdroplets, and (2) higher electrolyte

100 500m/z

(c)

(b)

(a)

[Cys+H]+

122.026 [Cys+Na]+

144.008

[Cys+2Na-H]+

165.990

0

100

[Cys-S-S-Cys+H]+

241.030

[Cys-S-S-Cys+Na]+

263.012

0

100

Rel

ativ

e A

bund

anc

e 122.026

144.008

165.990

Rel

ativ

e A

bund

anc

e

[Cys-S-S-Cys+2Na-H]+

284.994

243.046

Figure 2. (a) EC oxidation of cysteine (Cys). (b) Mass spec-trum showing P-EC/SSI-MS of Cys using ΔV = 4 V. (c) Massspectrum showing P-EC/SSI-MS of Cys when no voltage isapplied to the EC cell

Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry 1705

concentrations increase IE, which is in agreement with theproposed statistical charge distribution mechanism for SSI(average charge of the microdroplets is proportional to thesquare root of ion concentration) [83].

The optimum voltage for EC conversion of Cys was deter-mined by varying ΔV and monitoring product/reactant intensi-ty ratios, which increase from 2 to 4 V and then plateaus at 4 V(Figure 3c). These observations indicate that 2 V is the thresh-old for appearance of Cys-S-S-Cys ion signals in acquiredmassspectra ([Cys] = [KCl] = 10 mM) and that 4 V producesmaximum conversion under P-EC/SSI-MS conditions. Inci-dentally, [Cys] = 1 mM is the threshold concentration for

observing oxidation using P-EC/SSI-MS when ΔV = 4 V and[KCl] = 10 mM (Figure S1a). This voltage range (2 - 4 V)agrees with previous flow-EC/LS-DESI-MS oxidation of anal-ogous compound glutathione [10]. From the equation of thereaction (Figure 2a), 2 mol of Cys are converted for every molof disulfide product, which agrees with [Cys-S-S-Cys + H]+/[Cys + H]+ = 0.7 ± 0.1 for ΔV > 4 V ( plot in Figure 3c).Interestingly, Σ[Cys-S-S-Cys + n]+/Σ[Cys + n]+ = 1.2 ± 0.2 (plot in Figure 3c; the values for Σ[Cys + n]+ and Σ[Cys-S-S-Cys + n]+ can be obtained from the and plots in Figure 3b,respectively), suggesting that the disulfide product has highersodiation efficiency comparedwith Cys under these conditions.In addition, at ΔV = 4 V, the measured values for Σ[Cys + n]+,Σ[Cys-S-S-Cys + n]+, and Σ[Cys-S-S-Cys + n]+/Σ[Cys + n]+

increase from [KCl] = 1 to 10 mM and then plateaus from 10 to100 mM, indicating that [KCl] = 10 mM is the optimumconcentration for Cys oxidation without causing significant saltdeposition on the inlet capillary (see and plots in Figure 3band Figure S1c).

An important parameter in P-EC/SSI-MS is the polarity ofΔV with respect to direction of the microdroplet spray and MSinlet. All experiments described above were performed with E2connected to the positive terminal of the DC power supply(Figure 1a). When the polarity is switched so that E2 is thecathode, [Cys-S-S-Cys + n]+ ion signals were not observed(Figure S2). In addition, [Cys + n]+ signal intensities remainrelatively unchanged when E2 is the cathode. These resultssuggest that disulfide species generated at the anode (E1) arereduced at the cathode (E2) prior to exiting the EC assembly,producing mass spectra containing primarily [Cys + n]+

species.P-EC/SSI-MS characterization of the oxidation of dopamine

(DOP) to dopamine o-quinone (DOPQ) was used to estimate tr(Figure 4). Application of 4 V to the EC cell yielded massspectra containing ion signals at m/z 137.060 [DOP – NH3 +H]+, m/z 154.086 [DOP + H]+, m/z 176.068 [DOP + Na]+, m/z307.166 [2DOP + H]+, m/z 329.148 [2DOP + Na]+, and oxi-dation product at m/z 152.071 [DOPQ + H]+ (Figure 4b). Theextracted ion chromatogram (XIC) for [DOPQ + H]+ showsthat oxidation only occurs when a potential is applied (ONstate) to the EC cell. Now, tr = delay from voltage onset (delaytime) + time for signal intensity to increase to S/N = 3 (risetime). Signal intensity at the base (tbase) of the first peak is 295and at the point where the slope of the peak decreases is 3638(these points are connected by a red line, Figure 4c). Thus, risetime = 500(2 × 295)/(3638 – 295) = 90 ms (IT = 500 ms; totalscan time = 3 s). Similar calculations for the first three peaks inFigure 4c gives an average rise time of 120 ± 40ms (for peak 3,tbase is equal to the average noise intensity of ~30; note that theslope of peaks 3 and 4 decrease after two MS scans relative totbase). For these experiments, the temporal resolution using IT =500 ms is not sufficient to measure the delay time. ITMS XIC(IT = 100 ms for [PhSPh]•+ (m/z 186; reaction mechanismdiscussed later in the article) generated from EC oxidation ofPhSPh, yielded a rise time of 100 ± 100 ms and delay time of330 ± 10, which gives tr = 500 ± 100 ms (Figure 4d).

(c)

(b)

(a)

0

3

0 5 × 10-3 1 × 10-2

× 105

[M +

n]+

[Cys] in the Microdroplet Spray (M)

0

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0 5 × 10-2 1 × 10-1

× 105

V = 0 V (M = Cys)

V = 4 V (M = Cys)

V = 4 V (M = Cys-S-S-Cys)

[M +

n]+

[KCl] (M)

[Cys] in the Spray = 10 mM

No P-EC (M = Cys)

Planar P-EC (M = Cys; V = 0 V; No KCl)

0

8

0.0 2.0 4.0 6.0V (V)

[Cys

-S-S

-Cys

+ n

]+ /[C

ys +

n]+ n = Na

n = (2Na – H)n = Hn = H, Na, and (2Na – H)

[Cys

-S-S

-Cys

+ n

]+ /[C

ys +

n]+

[Cys] = [KCl] = 10 mM

Figure 3. Characterization of planar P-EC/MS. (a) Sum of theion signal intensities corresponding to Cys (Σ[M + n]+, where n =H, Na, and (2Na – H);M = Cys) as a function of [Cys] in the SSImicrodroplet spray. (b) Plot showing the influence of [KCl] onion signals corresponding to Cys and Cys-S-S-Cys. (c) Plotshowing the effect of EC potential on the signal intensities ofthe protonated, sodiated, and disodiated species of Cys (leftaxis). The plot also shows ΔV effect on the sum of all ion signalscorresponding to Cys and the disulfide product (right axis)

1706 Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry

In summary, experiments characterizing planar P-EC/SSI-MS show that (1) the paper substrate in the EC cell does notaffect ion signal intensity (viz. TE), (2) [electrolyte] influences IEowing to the square root dependence of average microdropletcharge on ion concentration, (3) the magnitude of ΔV applied tothe EC cell has no effect on IE or TE, (4) this P-EC cell config-uration has threshold [KCl] = 10mM and [Cys] = 1mM atΔV=4 V (threshold potential = 2 V) for observing disulfide product,(5) P-EC allows the use of relatively high [electrolyte] ≤ 10 mMwithout fouling of theMS inlet (deposition of KCl was observedat 100 mM), (6) the polarity of ΔV relative to the SSImicrodroplet spray direction and MS inlet determine the speciesobserved in acquired mass spectra (viz. the mass spectrometer

primarily samples species generated at E2 in the P-EC cell), and(g) the response time is on the order of hundreds of milliseconds.

Ionization and Differentiation of Polycyclic Aro-matic Hydrocarbons (Planar P-EC/SSI-MS)

It is often necessary to characterize samples containing nonpolarmolecules such as polycyclic aromatic hydrocarbons (PAHs),which are components of crude oil [84]. Many PAHs such as theisomers of benzo[α]pyrene (BAP) are potent carcinogens[33]that require environmental monitoring. Under certain conditions,ESI can extract an electron from PAHs when the number ofaromatic rings in themolecule is greater than two [84, 85]. Directdetection of PAHs by SSI-MS is more challenging because ahigh voltage is not applied to the microdroplet spray solution. Inaddition, MS is unable to resolve isomers, thus requiring addi-tional derivatization steps for characterization of PAH.

We utilized P-EC/SSI-MS to ionize and differentiate BAPand perylene (PER) isomers (C20H12; 252 Da) based on theintensities of species generated in EC reactions with H2O.When mixtures of BAP:PER (9 mM:1 mM and 1 mM:9 mMin CH2Cl2/ACN) were placed in the SSI microdroplet spray,[C20H12]

•+ (m/z 252.091) was not observed (Figure 5a and b),indicating that the SSI mechanism is not sufficient to removean electron from this aromatic ring system. This observationagrees with previous studies showing that the ESI potentialionizes PER with very low efficiency and does not yield anydetectable signal for BAP [33]. Application of ΔV = 4 V to theEC cell yielded ion signals at m/z 252.091 ([BAP]•+ and[PER]•+) for both the 9:1 and 1:9 solutions (Figure 5c and d).

Interestingly, a peak at m/z 267.078 is also observed, whichcorresponds to the formation of BAP and PER oxidation prod-ucts ([BAP – H + O]+ and [PER – H + O]+) from reaction withH2O (Figures 5c, d and e shows the proposed mechanism foroxidation of BAP). The intensity ofm/z 267.078 relative tom/z252.078 is as follows: 1 mM PER = ~5% (data not shown);9 mM BAP:1 mM PER > 100%; (Figure 5d); and, 1 mMBAP:9 mM PER = ~10% (Figure 5c). These results suggestthat EC oxidation of BAP has faster kinetics and/or [BAP –H+O] has higher ionization efficiency compared to [PER – H +O], which agrees with previous EC/ESI-MS analyses showingno [PER – H + O]+ in acquired mass spectra [33, 34, 54]. Thesignificant difference in the signal intensities of the oxidationproducts provides a means to distinguish a mixture of the twoisomers using EC/MS. Previous EC/ESI-MS spectra of H2O-mediated oxidation of BAP also show a peak at nominal m/z283 corresponding to [BAP – H + 2O]+ with higher relativeintensity compared with nominalm/z 267 and nominalm/z 252[33]. The ion signal at m/z 283.073 is observed at very lowrelative intensities of ~0.9%, ~0.3%, and ~0.4% (comparedwith m/z 252.091) for 1 mM PER, 1:9 BAP:PER, and 9:1BAP:PER solutions, respectively, when 4 V was applied to theEC cell (Figure 5c and d). The lower relative intensity (<1%) ofthe ion signal at nominalm/z 283 suggests that planar P-EC/SSI-MS (tr = 500 ± 100 ms) is accessing an earlier reaction timepoint compared with the EC/ESI-MS (tr < 3 s [33]). These

(c)

(b)

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Figure 4. Response time of planar P-EC/SSI-MS. (a) EC oxi-dation of dopamine (DOP) to formdopamine o-quinone (DOPQ).(b) Average Orbitrap mass spectrum across peak 1 in theextracted ion chromatogram (XIC) shown in (c). (c) XIC for[DOPQ + H]+ with (ON) and without voltage applied to the ECcell. Red lines indicate the rising edge of each peak. (d) ITMSXIC for [PhSPh]•+ (m/z 186; see Figure 6 for reaction mechanismand Orbitrap mass spectra; arrows indicate the time when theDC power supply is switched ON for each trial, which havedistinct colors)

Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry 1707

results highlight the ability of planar P-EC/SSI-MS to increaseIE, and sensitivity of nonpolar analytes, differentiate isomersbased on distinct EC properties and reactions, and interceptEC intermediates and products on short time scales.

Elucidation of Electrochemical Reaction Mecha-nisms (Planar P-EC/SSI-MS)

The characterization of EC reactions is important for under-standing many important chemical and biological processes[5]. One well-known reaction is the anodic oxidation of

diphenyl sulfide (PhSPh) to produce species containing sulf-oxide and sulfone functional groups (Figure 6a) [42]. Theproposed EC reaction mechanism [42] involves oxidation ofneutral [PhSPh] to yield radical cation [PhSPh]•+ (m/z 186.050),which is unstable and may undergo further oxidation to diphenylsulfone ([PhS(O)2Ph + H]+ at m/z 219.048 with ~0.1% relativeintensity compared with m/z 203.035) via diphenyl sulfoxide(observed as [PhS(O)Ph + H]+ at m/z 203.053, [PhS(O)Ph +Na]+ at m/z 225.035, and [2PhS(O)Ph + Na]+ at m/z 427.080)in the presence of H2O (Figure 6). Alternatively, [PhSPh]•+ mayreact to yield pseudodimer sulfonium ion [Ph2S

+C6H4SPh] atm/z371.093 and other oxidation products ([Ph2S

+C6H4S(O)Ph] atm/z 387.087 with ~0.3% relative intensity compared with m/z

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Figure 5. Planar P-EC/SSI-MS (electrolyte [KNO3] = 10 mM)spectra of solutions containing benzo[α]pyrene (BAP) andperylene (PER) in CH2Cl2/ACN. Mass spectra for (a) BAP:PER(1mM:9mM) and (b)BAP:PER (9mM:1mM) solutions usingΔV= 0 V (background signals shownwith red labels). Mass spectrafor (c) BAP:PER (1:9) and (d) BAP:PER (9:1) solutions using ΔV= 4 V. Chemical structures for BAP and PER are shown in panel(c). (e) EC oxidation of BAP in the presence of water

Figure 6. (a) Proposed mechanism for EC oxidation of[PhSPh] [42, 86]. (b) Planar P-EC/SSI-MS ([KCl = 10 mM]) of[PhSPh] (10 mM in ACN) using applied EC potential (ΔV) = 4 V.(c) Same as (b) using ΔV = 0 V

1708 Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry

203.035 and [Ph2S+C6H4S(O)2Ph], which was not observed). All

of these species, except [Ph2S+C6H4S(O)2Ph], were observed via

P-EC/SSI-MS (Figure 6b) including predominant backgroundions at nominalm/z 289, 301, and 317, which were also observedat ΔV = 0 V (Figure 6c).

Three mechanisms have been proposed for the formation ofthe pseudodimer sulfonium ion involving three distinct inter-mediates (bolded in the scheme below) [42, 86]:

Mechanism I

2 PhSPh½ �→2 PhSPh½ �•þ þ 2e−

m=z 186:050ð ÞPhSPh½ �•þ⇄ PhSC6H4½ �• þ Hþ

185:042 Dað ÞPhSPh½ �•þ þ PhSC6H4½ �•→ Ph2S

þC6H4SPh½ �m=z 371:093ð Þ

Mechanism II

PhSPh½ �→ PhSPh½ �•þ þ e−

PhSPh½ �•þ→ PhSC6H4½ �þ þ e− þ Hþ

m=z 185:042ð ÞPhSC6H4½ �þ þ PhSPh½ �→ Ph2S

þC6H4SPh½ �m=z 371:093ð Þ

Mechanism III

PhSPh½ � → PhSPh½ �•þ þe−PhSPh½ �•þ þ PhSPh½ �→ Ph2SC6H5SPh½ �•þ

m=z 372:101ð ÞPh2SC6H5SPh½ �•þ→ Ph2SC6H4SPh½ �þ þ e− þ Hþ

m=z 371:093ð Þ

Previous EC/ESI-MS studies (ΔV = 1.5 – 1.75 V) using tr =3 s, identified [PhSPh]•+ at nominal m/z 186 and thepseudodimer sulfonium ion at nominalm/z 371, but ion signalsfor the intermediates at nominal m/z 372 and 185 were notobserved, which led to the conclusion that mechanism I ispredominant [42]. Using P-EC/SSI-MS (tr = 500 ± 100 ms),an ion signal was observed at m/z 185.042 for intermediate[PhSC6H4]

+ indicating that mechanism II is also involved inthe formation of the pseudodimer sulfonium ion. The Orbitrapmass spectrometer used in this study is not capable of resolvingthe 13C isotope of pseudodimer sulfonium ions (m/z 372.096)and [Ph2SC6H5SPh]

•+ (m/z 372.101) (Figure 6b). However, therelative intensity of nominal m/z 372 (~26%) agrees with theexpected value for the 13C pseudodimer sulfonium isotope,suggesting that mechanism III is not predominant. These re-sults demonstrate that P-EC/SSI-MS can intercept intermedi-ates generated at electrode surfaces with temporal resolution ofhundreds of milliseconds, making it a powerful technique forelucidating fast EC processes.

Adding Reagents to the Paper Substrate in Planarand Tubular P-EC/SSI-MS

Addition of [PhSPh] and electrolyte to the paper substrate ofplanar P-EC/SSI-MS yieldedmore complexmass spectra havingthe same base peak ([PhS(O)Ph + H]+ at m/z 203.053) and withsignificantly lower relative intensities of relevant EC species(Figure 6a and mechanism II) compared with placing reagentin the microdroplet spray (compare Figure 6b and Figure S3a).The relative intensities of species relevant to anodic oxidation of[PhSPh] are 5% [PhSPh]•+ (m/z 186.050), 0.05% [PhSC6H4]

+

(m/z 185.042), 25% [PhS(O)Ph + Na]+ (m/z 225.035), 0.4%[Ph2S

+C6H4SPh] (m/z 371.093), 3% [2PhS(O)Ph + Na]+ (m/z427.080), and 1% [Ph2S

+C6H4S(O)Ph] at m/z 387.087 (viz. allspecies in Figure 6 and mechanism II observed except[PhS(O)2Ph+H]

+ at m/z 219.048). These results suggest thatplacing reagent in the paper causes unwanted competitivereaction pathways leading to numerous unidentified ionsignals in acquired mass spectra. These ion signals mayoriginate from additional [PhSPh]-related redox processesand/or from reaction with components of the papermatrix.

When tubular P-EC/SSI-MS (Figure 1b) was used to char-acterize oxidation of [PhSPh] in the microdroplet spray, ac-quired mass spectra (Figure S3b) contained many of the sameunidentified ion signals observed in planar P-EC/SSI-MS with[PhSPh] added to the paper electrolyte layer (Figure S3a). Inaddition, only the [PhS(O)Ph + H]+ (m/z 203.053), [PhS(O)Ph+ Na]+ (m/z 225.035), and [PhSPh]•+ (m/z 186.050) speciesrelevant to anodic [PhSPh] oxidation were observed. Impor-tantly, pseudodimer sulfonium ion was not observed and[PhS(O)Ph + H]+ was no longer the base peak (Figure S3b).When [PhSPh] was added to the paper in tubular P-EC/SSI-MS, ion signals for [Ph2S

+C6H4SPh] (m/z 371.093) and oxida-tion product [Ph2S

+C6H4S(O)Ph] (m/z 387.087) were alsoobserved, indicating EC conversion (Figure S3c). Thus, thefour configurations can be placed in order of ([PhSPh]oxidation):(unwanted competitive pathways) ratio as follows:planar-spray > planar-paper > tubular-paper > tubular-spray(EC cell shape-reagent location). This order suggests that com-petitive pathways are more pronounced at longer reactiontimes, which is controlled by the tube dimensions and reagentresidence time in the paper substrate.

Current results demonstrate that (1) reagents added to thepaper substrate undergo EC conversion with potential reactionwith components of the paper matrix and longer residencetimes, (2) EC transformation can be achieved using a tubularP-EC/SSI-MS design, and (3) tube dimensions significantlyimpact reactivity and performance characteristics such as ion-ization efficiency.

Proposed Mechanism for P-EC/SSI-MS

The results outlined above have provided some preliminaryinsights into the mechanism of P-EC/SSI-MS. When a voltageis applied to the EC cell, oxidation and reduction regionsextend a short distance from the electrode surfaces into the

Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry 1709

paper matrix. Microdroplets from the SSI emitter impact thepaper substrate, delivering reagents to the EC cell. Thesereagents may undergo reduction at E1 as they diffuse throughthe paper towards the anode E2, where they are oxidized.Owing to the thinness of the electrodes (200 μm) and paper(~100 μm), the speed of impacting microdroplets (~150 m/s[87]) rapidly transfers species generated at the E2-paper inter-face into the gas-phase, leading to a response time on thehundreds of milliseconds times scale (tr = 500 ± 100;Figure 4). The short tr allows detection of transient EC speciessuch as [PhSC6H4]

+ formed in the oxidation of [PhSPh] (Fig-ure 6). When reagents are added to the paper, there is a longertime between application of the voltage and sampling by themicrodroplet spray (viz. tr is longer), which may cause unwant-ed processes such as reaction with chemical components of thepaper. When the polarity is switched, reagents in themicrodroplet spray may be oxidized at E1 and then reducedat E2 (cathode) prior to entering the gas phase. As a result, ionsignals corresponding to oxidized intermediates and productswere not observed in positive mode mass spectra (Figure S2).For tubular P-EC/SSI-MS, tr should be longer because ofmultiple collisions with the paper as microdroplets travelthrough the tube. In addition, extraction efficiency from theinner surfaces of the tube will be lower compared with thedirect 90o impact of the microdroplet spray in the planarconfiguration. Similar to the planar configuration, the anodeshould be the inner electrode for characterization of oxidationreactions.

An important point to note is that relatively high concentra-tions of analyte (mM) were required to observe EC conversion.This concentration regime agrees with previous observationsby Cui et al. [3] that solid-contact K+-selective electrodes haddetection limits three orders of magnitude higher in a nitrocel-lulose paper matrix compared to bulk solution. A possiblereason for the lower sensitivity is that ion transport throughthe paper matrix and at the paper–electrode interface is slowercompared with solution phase. Further characterization exper-iments are required to identify the processes occurring in thepaper matrix and inside the tube during EC cell operation, aswell as elucidate their effect on EC reactivity. In addition, theimpact of parameters such as tube dimensions (e.g., length anddiameter), composition of the paper matrix, electrode material,and paper porosity on P-EC/MS performance needs to beevaluated.

ConclusionsThis report describes coupling of paper-based electrochemicalcells (P-EC) to sonic spray ionization mass spectrometry (P-EC/SSI-MS). The EC cells consist of a paper substratesandwiched between two mesh stainless steel electrodes. InP-EC/SSI-MS, the EC cell is physically decoupled from theionization source, allowing independent control of electricaland chemical parameters. The flexibility of paper and meshelectrodes allowed development of tubular and planar P-EC/

SSI-MS configurations, demonstrating high versatility andmodularity. P-EC/SSI-MS was used to successfully character-ize EC oxidation of cysteine, dopamine, polycyclic aromatichydrocarbons, and diphenyl sulfide. Perhaps one of the mostexciting features of P-EC/SSI-MS is a demonstrated responsetime on the order of hundreds of milliseconds, which allowedonline detection of transient EC species generated at the elec-trode surfaces in contact with the paper substrate. The hightemporal resolution makes P-EC/SSI-MS a powerful tool forelucidating the mechanisms of EC processes in situ. In addi-tion, the benefits and convenience of paper provide a low cost,disposable, and simple approach for coupling EC processes toMS. P-EC/MS has the potential to open new areas of researchand to facilitate the development of P-EC devices via advanc-ing current understanding of molecular EC mechanisms oper-ating in the paper substrate.

AcknowledgmentsThe Perry Research Laboratory gratefully acknowledges finan-cial support from the University of Illinois at Urbana-Champaign (UIUC).

References1. Cate, D.M., Adkins, J.A., Mettakoonpitak, J., Henry, C.S.: Recent devel-

opments in paper-based microfluidic devices. Anal. Chem. 87, 19–41(2015)

2. Dungchai, W., Chailapakul, O., Henry, C.S.: Electrochemical detection forpaper-based microfluidics. Anal. Chem. 81, 5821–5826 (2009)

3. Cui, J., Lisak, G., Strzalkowska, S., Bobacka, J.: Potentiometric sensingutilizing paper-based microfluidic sampling. Analyst 139, 2133–2136(2014)

4. Bruckenstein, S., Raogadde, R.: Use of a porous electrode for in-situ massspectrometric determination of volatile electrode reaction products. J. Am.Chem. Soc. 93, 793–794 (1971)

5. Liu, P.Y., Lu, M., Zheng, Q.L., Zhang, Y., Dewald, H.D., Chen, H.: Recentadvances of electrochemical mass spectrometry. Analyst 138, 5519–5539(2013)

6. Faber, H., Vogel, M., Karst, U.: Electrochemistry/mass spectrometry as atool in metabolism studies: a review. Anal. Chim. Acta 834, 9–21 (2014)

7. Permentier, H.P., Bruins, A.P.: Electrochemical oxidation and cleavage ofproteins with on-line mass spectrometric detection: Development of aninstrumental alternative to enzymatic protein digestion. J. Am. Soc. MassSpectrom. 15, 1707–1716 (2004)

8. Shumyantseva, V.V., Suprun, E.V., Bulko, T.V., Archakov, A.I.: Electro-chemical methods for detection of post-translational modifications of pro-teins. Biosens. Bioelectron. 61, 131–139 (2014)

9. Permentier, H.P., Bruins, A.P., Bischoff, R.: Electrochemistry-mass spec-trometry in drug metabolism and protein research. Mini-Rev. Med. Chem.8, 46–56 (2008)

10. Li, J., Dewald, H.D., Chen, H.: Online Coupling of electrochemical reac-tions with liquid sample desorption electrospray ionization-mass spectrom-etry. Anal. Chem. 81, 9716–9722 (2009)

11. Zhang, Y., Dewald, H.D., Chen, H.: Online mass spectrometric analysis ofproteins/peptides following electrolytic cleavage of disulfide bonds. J.Proteome Res. 10, 1293–1304 (2011)

12. McClintock, C., Kertesz, V., Hettich, R.L.: Development of an electro-chemical oxidation method for probing higher order protein structure withmass spectrometry. Anal. Chem. 80, 3304–3317 (2008)

13. Baumann, A., Lohmann, W., Jahn, S., Karst, U.: On-line electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) for the Generationand identification of nucleotide oxidation products. Electroanalysis 22,286–292 (2010)

1710 Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry

14. Jahn, S., Karst, U.: Electrochemistry coupled to (liquid chromatography/)mass spectrometry-current state and future perspectives. J. Chromatogr. A1259, 16–49 (2012)

15. Xu, K., Zhang, Y., Tang, B., Laskin, J., Roach, P. J., Chen, H.: Study ofhighly selective and efficient thiol derivatization using selenium reagents bymass spectrometry. Anal. Chem. 82, 6926–6932 (2010)

16. Wang, Z., Zhang, Y., Zhang, H., Harrington P., Chen, H.: Fast and selectivemodification of thiol proteins/peptides by N-(phenylseleno)phthalimide. J.Am. Soc. Mass Spectrom. 23, 520–529 (2012)

17. Modestov, A.D., Srebnik, S., Lev, O., Gun, J.: Scanning capillarymicroscopy/mass spectrometry formapping spatial electrochemical activityof electrodes. Anal. Chem. 73, 4229–4240 (2001)

18. Momotenko, D., Qiao, L., Cortes-Salazar, F., Lesch, A., Wittstock, G.,Girault, H.H.: Electrochemical push-pull scanner with mass spectrometrydetection. Anal. Chem. 84, 6630–6637 (2012)

19. Gun, J., Bharathi, S., Gutkin, V., Rizkov, D., Voloshenko, A., Shelkov, R.,Sladkevich, S., Kyi, N., Rona, M., Wolanov, Y., Rizkov, D., Koch, M.,Mizrahi, S., Pridkhochenko, P.V., Modestov, A., Lev, O.: Highlights incoupled electrochemical flow cell-mass spectrometry, EC/MS. Isr. J. Chem.50, 360–373 (2010)

20. Fenn, J.B., Mann, M., Meng, C.K., Wong, S.F., Whitehouse, C.M.:Electrospray Ionization for mass-spectrometry of large biomolecules. Sci-ence 246, 64–71 (1989)

21. Yamashita, M., Fenn, J.B.: Electrospray ion-source—another variation onthe free-jet theme. J. Phys. Chem. 88, 4451–4459 (1984)

22. Yamashita, M., Fenn, J.B.: Negative-ion production with the electrosprayion-source. J. Phys. Chem. 88, 4671–4675 (1984)

23. Blades, A.T., Ikonomou, M.G., Kebarle, P.: Mechanism of electrospraymass-spectrometry—electrospray as an electrolysis cell. Anal. Chem. 63,2109–2114 (1991)

24. Zeleny, J.: The electrical discharge from liquid points, and a hydrostaticmethod of measuring the electric intensity at their surfaces. Phys. Rev. 3,69–91 (1914)

25. Taylor, G.: Disintegration of water drops in electric field. Proc. Royal Soc.Lond. A-Mat. 280, 383–397 (1964)

26. Taylor, G.: Electrically driven jets. Proc. Royal Soc. Lond. A-Mat. 313,453–475 (1969)

27. Rohner, T.C., Lion, N., Girault, H.H.: Electrochemical and theoreticalaspects of electrospray ionization. Phys. Chem. Chem. Phys. 6, 3056–3068 (2004)

28. Van Berkel, G.J., McLuckey, S.A., Glish, G.L.: Electrochemical origin ofradical cations observed in electrospray ionization mass spectra. Anal.Chem. 64, 1586–1593 (1992)

29. Van Berkel, G.J., Zhou, F.M.: Electrospray as a controlled current electro-lytic cell—electrochemical ionization of neutral analytes for detection byelectrospray mass-spectrometry. Anal. Chem. 67, 3958–3964 (1995)

30. Van Berkel, G.J., Zhou, F.M.: Characterization of an electrospray ion-source as a controlled-current electrolytic cell. Anal. Chem. 67, 2916–2923 (1995)

31. Van Berkel, G.J., Zhou, F.M., Aronson, J.T.: Changes in bulk solution pHcaused by the inherent controlled-current electrolytic process of anelectrospray ion source. Int. J. Mass Spectrom. 162, 55–67 (1997)

32. Bond, A.M., Colton, R., D'Agostino, A., Downaed, A.J., Traeger, J.C.: Arole for electrospray mass spectrometry in electrochemical studies. Anal.Chem. 67, 1691–1695 (1995)

33. Xu, X., Lu, W., Cole, R.B.: On-line probe for fast electrochemistry/electrospray mass spectrometry. investigation of polycyclic aromatic hy-drocarbons. Anal. Chem. 68, 4244–4253 (1996)

34. Zhou, F., Van Berkel, G.: Electrochemistry combined on-line withelectrospray mass spectrometry. Anal. Chem. 67, 3643–3649 (1995)

35. Oberacher, H., Pitterl, F., Erb, R., Plattner, S.: Mass spectrometric methodsfor monitoring redox processes in electrochemical cells. Mass Spectrom.Rev. 34, 64–92 (2015)

36. Zhang, T.Y., Palii, S.P., Eyler, J.R., Brajter-Toth, A.: Enhancement ofionization efficiency by electrochemical reaction products in on-lineelectrochernistry/electrospray ionization Fourier transform ion cyclotronresonance mass spectrometry. Anal. Chem. 74, 1097–1103 (2002)

37. Looi, D.W., Eyler, J.R., Brajter-Toth, A.: Electrochemistry-electrosprayionization FT ICR mass spectrometry (EC ESI MS) of guanine-tyrosineand guanine-glutathione crosslinks formed on-line. Electrochim. Acta 56,2633–2640 (2011)

38. Mautjana, N.A., Estes, J., Eyler, J.R., Brajter-Toth, A.: One-electron oxi-dation and sensitivity of uric acid in on-line electrochemistry and in

electrospray ionization mass spectrometry. Electroanalysis 20, 2501–2508(2008)

39. Mautjana, N.A., Estes, J., Eyler, J.R., Brajter-Toth, A.: Antioxidant path-ways and one-electron oxidation of dopamine and cysteine in electrosprayand on-line electrochemistry electrospray ionization mass spectrometry.Electroanalysis 20, 1959–1967 (2008)

40. Mautjana, N.A., Looi, D.W., Eyler, J.R., Brajter-Toth, A.: Sensitivity ofPositive Ion Mode Electrospray Ionization Mass Spectrometry (Esi Ms) inthe Analysis of Purine Bases in Esi Ms and on-Line Electrochemistry EsiMs (Ec/Esi Ms). Electrochim. Acta 55, 52–58 (2009)

41. Mautjana, N.A., Looi, D.W., Eyler, J.R., Brajter-Toth, A.: Sensitivity ofPositive Ion Mode Electrospray Ionization Mass Spectrometry in the Anal-ysis of Thiol Metabolites. Electroanalysis 22, 79–89 (2010)

42. Lu, W., Xu, X., Cole, R.B.: On-line linear sweep voltammetry electrospraymass spectrometry. Anal. Chem. 69, 2478–2484 (1997)

43. Kertesz, V., Van Berkel, G.J.: Study and application of a controlled-potential electrochemistry-electrospray emitter for electrospray mass spec-trometry. Anal. Chem. 77, 4366–4373 (2005)

44. Van Berkel, G.J., Asano, K.G.: Controlling analyte electrochemistry in anelectrospray ion source with a three-electrode emitter cell. Anal. Chem. 76,1493–1499 (2004)

45. Albert, A., Shelley, J.T., Engelhard, C.: Plasma-based ambient desorption/ionization mass spectrometry: state-of-the-art in qualitative and quantitativeanalysis. Anal. Bioanal. Chem. 406, 6111–6127 (2014)

46. Awad, H., Khamis, M.M., El-Aneed, A.: Mass spectrometry, review of thebasics: ionization. Appl. Spectrosc. 50, 158–175 (2015)

47. Cooks, R.G., Ouyang, Z., Takats, Z., Wiseman, J.M.: Ambient massspectrometry. Science 311, 1566–1570 (2006)

48. Harris, G.A., Galhena, A.S., Fernandez, F.M.: Ambient Sampling/ionizationmass spectrometry: applications and current trends. Anal. Chem. 83, 4508–4538 (2011)

49. Ifa, D.R., Wu, C.P., Ouyang, Z., Cooks, R.G.: Desorption electrosprayionization and other ambient ionization methods: current progress andpreview. Analyst 135, 669–681 (2010)

50. Li, L.P., Feng, B.S., Yang, J.W., Chang, C.L., Bai, Y., Liu, H.: Applica-tions of ambient mass spectrometry in high-throughput screening. Analyst138, 3097–3103 (2013)

51. Monge, M.E., Harris, G.A., Dwivedi, P., Fernandez, F.M.: Mass spectrom-etry: recent advances in direct open air surface sampling/ionization. Chem.Rev. 113, 2269–2308 (2013)

52. Takats, Z., Wiseman, J.M., Gologan, B., Cooks, R.G.: Mass spectrometrysampling under ambient conditions with desorption electrospray ionization.Science 306, 471–473 (2004)

53. Venter, A., Nefliu, M., Cooks, R.G.: Ambient desorption ionization massspectrometry. TRAC-Trend. Anal. Chem. 27, 284–290 (2008)

54. Miao, Z., Chen, H.: Direct analysis of liquid samples by desorptionelectrospray ionization-mass spectrometry (DESI-MS). J. Am. Soc. MassSpectrom. 20, 10–19 (2009)

55. Liu, P.Y., Zhang, J., Ferguson, C.N., Chen, H., Loo, J.A.: Measuringprotein-ligand interactions using liquid sample desorption electrosprayionization mass spectrometry. Anal. Chem. 85, 11966–11972 (2013)

56. Ferguson, C.N., Benchaar, S.A., Miao, Z.X., Loo, J.A., Chen, H.: Directionization of large proteins and protein complexes by desorptionelectrospray ionization-mass spectrometry. Anal. Chem. 83, 6468–6473(2011)

57. Miao, Z.X., Chen, H., Liu, P.Y., Liu, Y.: Development of Submillisecondtime-resolved mass spectrometry using desorption electrospray ionization.Anal. Chem. 83, 3994–3997 (2011)

58. Miao, Z.X., Wu, S.Y., Chen, H.: The study of protein conformation insolution via direct sampling by desorption electrospray ionization massspectrometry. J. Am. Soc. Mass Spectrom. 21, 1730–1736 (2010)

59. Cai, Y., Liu, Y., Helmy, R., Chen, H.: Coupling of Ultrafast Lc with MassSpectrometry by DESI. J. Am. Soc. Mass Spectrom. 25, 1820–1823 (2014)

60. Cai, Y., Adams, D., Chen, H.: A new splitting method for both analyticaland preparative LC/MS. J. Am. Soc. Mass Spectrom. 25, 286–292 (2014)

61. Zhang, Y., Yuan, Z.Q., Dewald, H.D., Chen, H.: Coupling of liquidchromatography with mass spectrometry by desorption electrospray ioni-zation (DESI). Chem. Commun. 47, 4171–4173 (2011)

62. Zheng, Q.L., Zhang, H., Chen, H.: Integration of online digestion andelectrolytic reduction with mass spectrometry for rapid disulfide-containing protein structural analysis. Int. J. Mass Spectrom. 353, 84–92(2013)

Y.-M. Liu and R. H. Perry: Paper Electrochemistry/Mass Spectrometry 1711

63. Zhang, Y., Cui, W.D., Zhang, H., Dewald, H.D., Chen, H.:Electrochemistry-assisted top-down characterization of disulfide-containing proteins. Anal. Chem. 84, 3838–3842 (2012)

64. Lu, M., Wolff, C., Cui, W.D., Chen, H.: Investigation of some biologicallyrelevant redox reactions using electrochemical mass spectrometryinterfaced by desorption electrospray ionization. Anal. Bioanal. Chem.403, 355–365 (2012)

65. Brown, T.A., Chen, H., Zare, R.N.: Identification of fleeting electrochem-ical reaction intermediates using desorption electrospray ionization massspectrometry. J. Am. Chem. Soc. 137, 7274–7277 (2015)

66. Wang, H., Liu, J.J., Cooks, R.G., Ouyang, Z.: Paper spray for directanalysis of complex mixtures using mass spectrometry. Angew. Chem.-Int. Ed. 49, 877–880 (2010)

67. Liu, J.J., Wang, H., Manicke, N.E., Lin, J.M., Cooks, R.G., Ouyang, Z.:Development, characterization, and application of paper spray ionization.Anal. Chem. 82, 2463–2471 (2010)

68. Cooks, R.G., Manicke, N.E., Dill, A.L., Ifa, D.R., Eberlin, L.S., Costa,A.B., Wang, H., Huang, G.M., Zheng, O.Y.: New ionization methods andminiature mass spectrometers for biomedicine: desi imaging for cancerdiagnostics and paper spray ionization for therapeutic drug monitoring.Faraday Discussions 149, 247–267 (2011)

69. Pulliam, C.J., Bain, R.M.,Wiley, J.S., Ouyang, Z., Cooks, R.G.:Mass spectrom-etry in the home and garden. J. Am. Soc. Mass Spectrom. 26, 224–230 (2015)

70. Espy, R.D., Teunissen, S.F., Manicke, N.E., Ren, Y., Ouyang, Z., vanAsten, A., Cooks, R.G.: Paper spray and extraction spray mass spectrom-etry for the direct and simultaneous quantification of eight drugs of abuse inwhole blood. Anal. Chem. 86, 7712–7718 (2014)

71. Bag, S., Hendricks, P.I., Reynolds, J.C., Cooks, R.G.: Biogenic aldehydedetermination by reactive paper spray ionization mass spectrometry. Anal.Chim. Acta. 860, 37–42 (2015)

72. Bain, R.M., Pulliam, C.J., Yan, X., Moore, K.F., Muller, T., Cooks, R.G.:Mass spectrometry in organic synthesis: Claisen-Schmidt base-catalyzedcondensation and Hammett correlation of substituent effects. J. Chem.Educ. 91, 1985–1989 (2014)

73. Hamid, A.M., Wei, P., Jarmusch, A.K., Pirro, V., Cooks, R.G.: Discrimi-nation of candida species by paper spray mass spectrometry. Int. J. MassSpectrom. 378, 288–293 (2015)

74. Hamid, A.M., Jarmusch, A.K., Pirro, V., Pincus, D.H., Clay, B.G., Gervasi,G., Cooks, R.G.: Rapid discrimination of bacteria by paper spray massspectrometry. Anal. Chem. 86, 7500–7507 (2014)

75. Hirabayashi, A., Sakairi, M., Koizumi, H.: Sonic spray ionization methodfor atmospheric-pressure ionization mass spectrometry. Anal. Chem. 66,4557–4559 (1994)

76. Hardman, M., Makarov, A.A.: Interfacing the Orbitrap mass analyzer to anelectrospray ion source. Anal. Chem. 75, 1699–1705 (2003)

77. Hu, Q.Z., Noll, R.J., Li, H.Y., Makarov, A., Hardman, M., Cooks, R.G.:The Orbitrap: a new mass spectrometer. J. Mass Spectrom. 40, 430–443(2005)

78. Makarov, A.: Electrostatic axially harmonic Orbital trapping: a high-performance technique of mass analysis. Anal. Chem. 72, 1156–1162(2000)

79. Makarov, A., Denisov, E., Kholomeev, A., Baischun, W., Lange, O.,Strupat, K., Horning, S.: Performance evaluation of a hybrid linear iontrap/Orbitrap mass spectrometer. Anal. Chem. 78, 2113–2120 (2006)

80. Makarov, A., Denisov, E., Lange, O., Horning, S.: Dynamic range of massaccuracy in LTQ Orbitrap hybrid mass spectrometer. J. Am. Soc. MassSpectrom. 17, 977–982 (2006)

81. Perry, R.H., Cooks, R.G., Noll, R.J.: Orbitrap mass spectrometry: instru-mentation, ionmotion, and applications. Mass Spectrom. Rev. 27, 661–699(2008)

82. Wijeratne, A.B.., Yang, S.H., Armstrong, D.W., Schug, K.A.: Solventmolecules undergo homolytic cleavage and radical recombination process-es during negative-mode electrospray ionization: adduct formation withantimony(III)-tartrate dianion. Anal. Chem. 82, 5141–5146 (2010)

83. Dodd, E.E.: The statistics of liquid spray and dust electrification by thehopper and laby method. J. Appl. Phys. 24, 73–80 (1953)

84. Guricza, L.M., Schrader, W.: Electrospray ionization for determination ofnon-polar polyaromatic hydrocarbons and polyaromatic heterocycles inheavy crude oil asphaltenes. J. Mass Spectrom. 50, 549–557 (2015)

85. Miyabayashi, K., Naito, Y., Tsujimoto, K., Miyake, M.: Structure charac-terization of polyaromatic hydrocarbons in arabian mix vacuum residue byelectrospray ionization fourier transform ion cyclotron resonance massspectrometry. Int. J. Mass Spectrom. 235, 49–57 (2004)

86. Magno, F., Bontempe, G.: Electrochemical behavior of diphenyl sulfide inacetonitrile medium at a platinum-electrode. J. Electroanal. Chem. 36, 389–397 (1972)

87. Venter, A., Sojka, P.E., Cooks, R.G.: Droplet dynamics and ionizationmechanisms in desorption electrospray ionization mass spectrometry. Anal.Chem. 78, 8549–8555 (2006)

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